This invention relates generally to enzyme purification and/or isolation by employing the substrate specificity of an enzyme, and more specifically to the purification and/or isolation from complex mixtures of materials of trypsin and trypsin-like proteolytic enzymes which include: trypsin (E.C.3.4.4.4), carboxypeptidase B (E.C.3.4.2.2), papain (E.C.3.4.4.10), ficin (E.C.3.4.4.12), thrombin (E.C.3.4.4.13), plasmin (E.C.3.4.4.14), subtilopeptidase A(E.C.3.4.4.16), aspergillopeptidase A(E.C.3.4.4.17), streptococcus peptidase A(E.C.3.4.4.18), clostridiopeptidase B(E.C.3.4.4.20), bromelain (E.C.3.4.4.24), and urokinase (E.C.3.4.4.a). A trypsin-like enzyme is hereby defined as a hydrolytic enzyme belonging to the Enzyme Commission (E.C.) class 3.4.-.-. and having a specificity for peptide bonds as well as ester bonds on arginine and lysine residues (Report of the Commission on Enzymes of the International Union of Biochemistry, Pergamon Press, Oxford, (1961)).
Generally known procedures for enzyme isolation employed in the prior art involve an array of discrete techniques which are used in empirically determined combinations. These techniques depend for the most part on some gross molecular parameter of the enzyme of interest such as its solubility in salt solutions, for example, in ammonium sulfate solutions or water miscible organic solvents, for example, ethanol, or acetone, or its electrical properties, as used in electrophoretic processes, isoelectric precipitation or ion exchange chromatography. In addition, molecular size has been used to purify enzymes as in dialysis, in gel filtration chromatography, and in ultrafiltration. Selection of any of these methods, singularly or in combination, is made in the prior art to optimize purification and recovery of the enzyme. Trypsin has been purified from bovine pancreas, employing methods more specifically outlined in an article by M. Laskowski in Methods In Enzymology, Vol. II, page 26, 1955. In this method minced pancreas is extracted with dilute sulfuric acid and treated with ammonium sulfate in several discrete steps to remove ribonuclease and alpha-chymotrypsinogen. The pH of the resulting solution is adjusted to 3.0 and treated with ammonium sulfate to precipitate the trypsinogen. This material is then extracted with 0.4 saturated ammonium sulfate solution and re-precipitated with additional ammonium sulfate. After washing with an acidified magnesium sulfate solution, the crude trypsinogen is dissolved in a pH 8 buffer containing CaCl.sub.2 and stored for 24 hours at 4.degree. C. The trypsin so obtained is then purified by further ammonium sulfate precipitations and extractions. This procedure is an example of purifying trypsin by solution-property methods.
Another method employed in the prior art is reported by Feinstein (FEBS Letters, 7: 353 [1970]). In this method trypsin is purified by adsorption onto an agarose column which contains covalently bound ovomucoid, a trypsin inhibiting protein obtained from chicken egg. The catalytically active trypsin is separated from non-active trypsin as well as from alpha-chymotrypsin. This technique is also referred to as enzyme-specific chromatography by Baker (Design of Active-Site-Directed Irreversible Enzyme Inhibitors, Wiley, N. Y. 1967) as well as affinity chromatography by Cuatrecasas, et al. (Proc. Natl. Acad. Sci., U.S., 61:636 [1968]). These methods differ from previously established enzyme purification methods in that the biocatalytic specificity of the enzyme is the means employed to achieve the purification.
Small molecule inhibitors in addition to the macromolecular inhibitors of trypsin and trypsin-like enzymes have been taught (Vogel, et al., Natural Proteinase Inhibitors, Academic, N. Y., 1968). Alkyl and aryl guanidines and amidines are known to be inhibitors of this type (Mares-Guia and Shaw, J. Biol. Chem. 240: 1579 (1965)).
Although the solution-property techniques have been employed and are currently employed to purify trypsin and trypsin-like enzymes among others, they have been proven to be inefficient in operation, expensive and time consuming. While the affinity method of Feinstein represents a simplification over solution-property techniques of enzyme purification, the use of macromolecular proteinase inhibitors has the following deficiencies. The macromolecular inhibitors have a broad enzyme specificity, furthermore these inhibitors must carefully be purified before they can be used in affinity matrices. Also, the concentration of these inhibitors in the affinity matrix is not easily controlled. Hence, there has been demonstrated a continuing need to provide simpler materials and more efficient methods to obtain these enzymes in higher yield and higher purity than heretofore possible.